The Triplet Expansion Diseases (TREDs) are a group of genetic disorders that result from an expansion in the size of a triplet repeat array in the coding or regulatory region of the affected gene. These disorders include Fragile X syndrome, the most common heritable form of mental retardation, and Friedreichs ataxia, the most common recessive ataxia. We are interested in both the mechanism of expansion and the consequences of expansion in these two disorders. We and others have shown that these repeats form a variety of intrastrand structures, and that the ability to form such structures is a common property of hypervariable sequences. It has been suggested that these structures contribute both to the initial expansion event and to its sequelae in a variety of ways. We have previously shown that alleles of the human FMR-1 gene that have a ~100% likelihood of expansion when maternally inherited in families with Fragile X syndrome, are not intrinsically unstable in transgenic mice. This suggests that factors other than the sequence itself are important for expansion. Genetic data in humans and instability studies of triplet repeats in bacteria and yeast from a number of laboratories including our own, suggest that various cis- and trans-acting factors may be important. These factors could include those that might permit secondary structures to form or that exacerbate their effects. To identify those cis-acting factors important in mammals we are in the process of generating mice containing long CGG- repeat tracts with different flanking sequences including a targeted insertion of 180 CGG-repeats into the murine FMR-1 gene. Potential trans-acting factors identified in bacteria or yeast include proteins in various DNA replication, surveillance or damage repair pathways. We are currently breeding our mice with mice containing mutations in the murine homologs of some of these genes. Work to date suggests that the presence or absence of the DNA damage surveillance protein p53 has no effect on CGG-repeat stability even under conditions that result in DNA damage. Work on the normal FMR-1 gene has allowed us to identify a variety of factors that are important for its regulation. In the course of this work we have identified a novel DNA binding protein as well as a novel RNA binding protein protein that may be involved in posttranscriptional regulation of FMR-1. We have also shown that one of the consequences of the absence of FMRP, the FMR-1 gene product, is a change in the levels of a number of important nuclear proteins. This may provide insight as to how the reduced levels of FMRP in patients with Fragile X syndrome leads to mental retardation. In vitro work on the consequences of the GAA-repeat expansion seen in Friedreichs ataxia has revealed an unusual, and potentially widespread means of transcriptional regulation, and also suggests a mechanism whereby this unusual sequence may cause expansion. We have shown in vitro that a triple-stranded DNA structure forms during transcription that traps the RNA polymerase. We have extended this work to demonstrate that this method of regulation operates in a simple in vivo model system. - Triplet expansion diseases,Mouse model,Fragile X syndrome, Friedreich's ataxia